The research work supported by this grant has changed its direction so that the major effort is now directed toward yeast mitochondrial development. This will be approached in three separate ways: (1) To directly investigate the gene products coded for by the yeast mitochondrial genome, purified mitochondrial DNA (mDNA) will be transcribed and translated in a coupled system and the protein products analyzed by gel electrophoresis and/or immunoprecipitation. (2) Nuclear gene mutants controlling mitochondrial development and behavior will be isolated by using techniques that selectively incorporate bromuracil (BU) into the DNA at an elevated temperature; temperature sensitive (ts) nuclear mutants that fail to replicate MDNA at the non- permissive temperature will be selected by their ability to survive when the cells containing BU labelled mDNA are irradiated with UV light at 300-400 nm. (3) An intensive search will be continued for the isolation of ts respiratory mutants that are mitochondrially inherited. This will be done by nitrosoguanidine (NS) co-mutagenesis that will allow one to clone out those mitochondria that have become resistant to an antibiotic due to a mitochondrial mutation and whose neighboring genes have simultaneously become mutagenized by NG to give rise to a ts gene product. Selection of cells resistant to the antibiotic at permissive temperatures will allow one to examine those cells where the mitochondrial genome also carries a ts missense mutation.